Comprehensive Pre-clinical Assessment of Microbicide Safety Using in vitro and Murine Models
Pedro Mesquita*, S Wilson, N Cheshenko, M Keller, B Galen, and B Herold
Albert Einstein Coll of Med, Bronx, NY, US
Background: The lack of biomarkers predictive of
efficacy and safety is a critical gap in microbicide development. This was
highlighted in a recent cellulose sulfate clinical trial, where treatment with
the microbicide was associated with an increased risk of HIV acquisition.
Measuring cytotoxicity in vitro does not suffice in terms of safety
assessment of candidate compounds. The multilayered epithelium in the vagina
and ectocervix provides the first line of defense against HIV and other
pathogens and disruption of this barrier facilitates HIV transmission. We
evaluated the effect of candidate microbicides on the architecture of polarized
epithelial cell cultures and murine genital tract tissue; expression of immune
mediators and the consequences for transmission of HIV.
Methods: Human epithelial cells or reconstituted
tissue were grown under polarized conditions in a dual chamber culture system.
Following in vitro treatment of cultured cells or intravaginal
application to the mouse genital tract (single or repeated) of cellulose sulfate,
PRO 2000, or tenofovir, the effect on epithelial cell integrity and cellular
junctions was evaluated by monitoring transepithelial electrical resistance
(TER) and by confocal microscopy. Furthermore, changes in expression of
structural proteins were detected at the RNA and protein levels. Finally, HIV addition
to the apical chambers in vitro allowed assessment of virus migration
across the epithelium to infect susceptible T cells cultured in the basolateral
Results: Application of cellulose sulfate (100 µg/mL
or 6% gel) led to a significant disruption of tight and adherens junctions in
both models, inducing a rapid and sustained loss of TER across epithelial cells.
The cellulose sulfate-induced disruption facilitated translocation of HIV
across the epithelium as evidenced by confocal microscopy. In contrast, tenofovir
had no deleterious effects. PRO 2000 caused a more modest loss in TER, but had
no effect on adherens junction proteins and no increase in HIV translocation
was observed. Repeated exposures to cellulose sulfate also triggered an
inflammatory response characterized by the release of inflammatory cytokines
and a loss of the protective protein secretory leukocyte protease inhibitor.
Conclusions: Together, these findings provide a
biological explanation that may have contributed to the failed cellulose sulfate
clinical trial and indicate tenofovir safety. These data suggest the inclusion
of the models presented in the preclinical evaluation of candidate